This invention, in the fields of immunology, biochemistry and cell and molecular biology, relates to proteins or proteins that are co- and/or post-translationally modified, termed LAIT proteins, that activate B cells. This invention is also directed to the use of such protein in pharmaceutical preparations, and pharmaceutical compositions comprising LAIT protein or functional derivatives thereof. This invention is also directed to nucleic acid molecules encoding the bovine LAIT protein or functional derivatives thereof and methods for the purification of native and recombinant forms of said proteins that activate B cells.
Bone marrow-derived xe2x80x9cBxe2x80x9d lymphocytes, commonly called B cells, are a type of white blood cell present in the lymph, the blood, and in secondary lymphoid organs of the immune system. B cells are the precursors of antibody secreting cells, plasma cells, and as such are central to the induction of humoral immune responses.
The induction of most humoral immune responses in the adult involves a number of cellular interactions among thymus-derived T lymphocytes, commonly called T cells, antigen presenting cells (APC), and B cells [J. Exp. Med 147:1159, 1978; PNAS 77:1612, 1982; PNAS 79:1989, 1982; Immunol. Rev. 95:914, 1987].
As currently understood, T cell-dependent B cell activation involves activation of T cells upon their recognition of antigen, as presented by APC in conjunction with proteins encoded within the major histocompatibility complex (MHC), which are expressed on the cell surface of the APC. This antigen specific and MHC restricted T cell-APC interaction results in reciprocal activation of the two cell types, and the alteration of T cell physiology such that xe2x80x9chelper functionxe2x80x9d becomes manifest.
Helper T cells can activate antigen specific B cells. Antigen specificity of the T cell-B cell interaction is maintained as a consequence of the ultimate capacity of the B cell to function as an APC. Thus, while resting, quiescent B cells are not efficient APC (PNAS 79: 1989, 1982), they specifically interact with antigen through membrane associated immunoglobulin, the specificity of which reflects that of the immunoglobulin their daughter cells will secrete (J. Exp. Med. 140:904, 1974).
Immunoglobulin mediated internalization of antigen by the specific B cell, which may involve presentation by yet another sort of APC, the follicular dendritic cell, results in the initiation of antigen processing by the B cell, the up-regulation of MHC Class II and B7 expressior., and the presentation of antigen derived peptides in the context of MHC (J. Exp. Med. 178: 2055, 1993). The B cell activated by this route is a target for the activated helper T cell.
T cell helper function includes signals delivered through both T cell-B cell contact, and the interaction of T cell derived soluble mediators, referred to as cytokines, with their cognate ligands expressed on the B cell plasma membrane. T cell-B cell contact is also MHC restricted, analogous to the T cell-APC interaction (Eur. J. Immunol. 12:627, 1982; Eur. J. Immunol. 12:634, 1982). However, the specific interaction of the molecules which mediate the MHC restricted interaction between the two lymphocyte lineages, specifically, the T cell receptor for antigen (TcR), and the MHC/antigen complex expressed by the B cells, do not predicate the induction of B cell growth and differentiation (Eur. J. Immunol. 18:375, 1988).
The essential molecular interaction, reflected by the requirement for T cell-B cell contact, is mediated by CD40 expressed on the plasma membrane of the B cell, and its cognate ligand, gp39 (or CD40L), expressed on the plasma membrane of the T cell (PNAS 89:6550, 1992; Nature 357:80, 1992). Consistent with this paradigm is the observation that membrane expression of the latter increases upon T cell-APC interaction, as well as subsequent to T cell-B cell interaction (PNAS 89:6550, 1992). Further, membrane immunoglobulin mediated B cell interaction with antigen results in the increased membrane expression of CD40 (Sem. in Immunol 6:303, 1994). The interaction between CD40 and CD40L predicates the induction of B cell growth, B cell differentiation into immunoglobulin secreting cells, and immunoglobulin isotype switching (J. Exp. Med. 178:1567, 1993).
Consistent with this model is the observation that soluble CD40L, or monoclonal antibody (mAb) specific for CD40 can induce B cell growth and differentiation to immunoglobulin secretion (Sem. in Immunol. 6:267, 1994; PNAS 83:4494, 1986; J. Immunol. 140:1425, 1988;).
In addition to the obligate requirement for T cell-B cell contact, a number of T cell derived cytokines, IL-2, IL-4 and IL-5 are central to B cell growth and differentiation. B cell susceptibility to these cytokines is for the most part limited by prior contact with a T cell. Thus, subsequent to T cell contact, the B cells increase expression of cytokine specific membrane receptors (PNAS 80:6628, 1983; J. Immunol. 145:2025, 1990; J. Immunol. 146:1118, 1991). IL-2 and IL-5 have been demonstrated to support the growth of activated B cells (PNAS 77:1612, 1980; Immunol. Rev. 52;115, 1980). Further, IL-4 and anti-immunoglobulin have been shown to synergize in supporting B cell growth (J. Exp. Med. 155:914, 1982).
Notable exceptions in this context are the quiescent B cell responses to IL-4 and IL-5. IL-4 induces the de novo transcription and translation of MHC Class II proteins (J. Exp. Med. 155:914, 1982; PNAS 81:6149, 1984; J. Exp. Med. 160;679, 1984), and IL-5 is able to support the differentiation of quiescent B cells into high rate immunoglobulin secreting cells in the absence of cell growth (Eur. J. Immunol. 22:2323, 1992).
In any event, signals derived from molecular interactions amongst membrane molecules on T cells and B cells, and from those of T cell derived cytokines interacting with their cognate receptors on B cells are parts of a complex signaling system. Each signal drives the B cell to another stage of activation, rendering it susceptible to subsequent progression signals. These signals complement one another, rather than having the capacity, individually, to drive the complete process of B cell growth and differentiation (Immunol. Rev. 95:177, 1987).
In 1988, a unique activity in ovine colostrum was discovered (J. Immunol. 140:1366, 1988). Proline Rich Protein (PRP) had been partially purified using classical techniques of protein purification. This material was shown to support the induction of quiescent B cells into the cell cycle, and to support their differentiation into high rate immunoglobulin secreting cells. This was apparently the first report of a protein of mammalian origin that mediates these functions.
A monoclonal antibody specific for ovine PRP was subsequently prepared. When PRP preparations were passed over an affinity column prepared using the antibody, all of the PRP was retained by the column, as assessed by Western blotting analysis of eluate and effluent. However, all of the B cell stimulatory activity was found in the effluent. Thus, the published characterization of the B cell tropic bioactivity present in ovine colostrum was not attributable to PRP (unpublished information).
This invention features a novel bovine protein and isolated nucleotide sequences encoding the protein, the said protein being capable of activating B-cells of mammalian origin. A substantially pure LAIT protein or co- and/or post-translationally modifiel form of the protein may be produced by biochemical purification, or by recombinant means in a prokaryotic or eukaryotic host substantially free of other proteins with which it is natively associated. Also included in this invention is a process for purifying LAIT protein or a co- and/or post-translationally modified form of LAIT protein of this invention from bovine colostral whey comprising:
(i) salting out of proteins contained within said samples
(ii) enrichment and ultimate purification of LAIT protein from proteins salted out in step (i) utilizing classical protein fractionation techniques.
In all cases the said protein possesses the desired biological activity.
The invention is also directed to a nucleic acid molecule comprising a nucleotide sequence encoding a LAIT protein. The nucleic acid molecule may be cDNA or genomic DNA.
The isolated bovine protein has homology with human CD14 and murine CD14 and so is also referred to as bovine CD14. The invention includes a method of activating B cells, and particularly of activating B cells in a mammal in need of such activation by administering CD14, a recombinant form of the protein thereof, or a functional derivative thereof.
In a preferred embodiment, the mammal is a human patient.
According to one aspect, the invention includes incorporating CD14 into infant formula. The invention includes administering CD14 to an infant, a preferred mode of administration being feeding to the infant such formula.
In another aspect, the invention includes incorporating CD14 as part of a vaccination. The invention includes administering CD14 and antigen to a patient in need of immunization, a preferred mode of administration including administering a single preparation containing both CD14 and the antigen.
In another aspect, the invention includes administering CD14 to a patient having a T cell immune deficiency. In a preferred aspect, the invention includes administering CD14 to a patient suffering from a particular T cell dysfunction in which gp39 (CD40L) is under expressed on or totally absent from the cell surface of patient T cells.
In another aspect, the invention includes administering antibodies raised against CD14 to a patient suffering from a dysfunction wherein the patient""s B cells are hyperactivated as a result of higher than normal levels of serum CD14. In a preferred aspect, the invention includes administering antibodies against CD14 to a patient suffering from rheumatoid arthritis wherein the B cells are secreting rheumatoid factor as a result of being activated by serum CD14.
This invention includes a novel method of the production of hybridomas secreting mAb of desired specificity by cuturing B cells with sub-optimal mitogenic concentrations of CD14 in concert with the antigen to which antibodies wished to be raised against. Populations of B cells activated in this manner are highly enriched for activated, antigen specific B cells, which are then be used for the production of hybridomas secreting the mAb of desired specificity.
The invention includes use of CD14 in preparation of medicaments for activating B cells in a mammal in need of such activation.
Natural or recombinant CD14 can be used in the invention.
In the context of this invention, the term xe2x80x9cCD14xe2x80x9d includes murine, bovine or human CD14.
A xe2x80x9cfunctional derivativexe2x80x9d retains at least a portion of the function of CD14, such as binding to a specific antibody or binding to its cognate receptor on cells that possess said receptor which permits its utility in accordance with the present invention. The term xe2x80x9cfunctional derivativexe2x80x9d as used herein includes a xe2x80x9cfragment,xe2x80x9d or xe2x80x9cvariantxe2x80x9d of CD14, which terms are defined below.
A xe2x80x9cfragmentxe2x80x9d of CD14 refers to any subset of the polypeptide, that is, a shorter peptide. The term xe2x80x9cfragmentxe2x80x9d is used to indicate a polypeptide which is derived from CD14 having a naturally occuring protein sequence comprising a deletion of one or more amino acids at one or more sites of the C-terminal, N-terminal, and within the sequence. Such fragments should retain one or more biological activities or functions which are characteristic for the intact CD14 polypeptide or co- and/or post-translationally modified forms of CD14.
A xe2x80x9cvariantxe2x80x9d of CD14 refers to a polypeptide having a primary sequence similar to that of the native CD14 or fragment thereof such that native activity is at least partially retained. Variant peptides may be prepared by synthetic means or by mutations in the cDNA encoding said polypeptide that retains biological activity of said polypeptide including deletions, insertions or conservative amino acid substitutions within the polypeptide.
The term xe2x80x9cantibodyxe2x80x9d as used herein is an immunoglobulin protein that has the capability to bind a distinct epitope in an unconserved region of said protein thereby enabling the antibody to distinguish one protein from another. The term xe2x80x9cepitopexe2x80x9d is meant to refer to that portion of any molecule capable of being bound by an antibody. The term xe2x80x9cantibodyxe2x80x9d includes polyclonal antibodies, monoclonal antibodies (mAbs) or chimeric antibodies.
Polyclonal antibodies are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen.
Monoclonal antibodies are a homogenous population of antibodies capable of binding a distinct epitope on the antigen. MAbs may be obtained by methods known to those skilled in the art. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The term xe2x80x9cantibodyxe2x80x9d is also meant to include both intact molecules as well as fragments thereof, such as, for example Fab and F(abxe2x80x2)2, which are capable of binding antigen. Fab and F(abxe2x80x2)2, lack the Fc fragment of the intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325, 1983).
Chimeric antibodies are molecules, different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a constant region derived from a human immunoglobulin.
An xe2x80x9cantigenxe2x80x9d is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. When an antibody is said to be xe2x80x9cspecific forxe2x80x9d a polypeptide, fragment, or variant thereof or is said to be xe2x80x9ccapable of bindingxe2x80x9d to a polypeptide, fragment, or variant thereof it is meant that the antigen will react in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.